The preparation of samples usually is the first step of many bioanalytical protocols on real samples, such as blood, food, or environmental samples. Often, a particular species of biomolecules is isolated from a mixture of other molecules and particle constituents and transferred into a new buffer. The invention described here concerns the centrifugal separation of particles from a suspension in which particles are suspended in a liquid.
Separating particles from suspensions by means of centrifuges represents an established standard method in laboratory analyses. Particles in suspension, such as the cellular proportion of blood, sediment under the influence of the centrifugal gravitational field according to their distribution of density. The particle suspension, for example the blood, is introduced into a rotating reservoir 10 (FIG. 1), which is rotatable about a rotation center 12. During rotation, for example at an angular velocity ω, a stack of three phases forms in the reservoir 10, as shown in FIG. 1. A particle-free supernatant liquid forms radially inwardly, whereas at the radially outward edge a phase of sedimented particles 16 forms due to its mass density, which is increased as opposed to the liquid. In the sedimentation, this outer phase 16 mainly represents a sediment of more dense, cellular blood constituents, consisting of cells. Between the particle-free supernatant 14 and the sediment, there initially still is a phase 18 consisting of particles in suspension. After a certain time, the phase 18 transitions into the particle-free supernatant 14 with its liquid proportion and into the sediment 16 with its particle proportion.
The complete transition is reached after a time t1, wherein at this time instant an equilibrium state is present in that particles and solution are separated from each other as far as possible, except for some liquid residue in the spaces between the sediment particles. As shown in FIG. 1, the interfaces between particle-free supernatant 14 and suspension 18, as well as between suspension 18 and sedimented particles 16 converge until the equilibrium is finally reached at the time instant t1. In FIG. 1, the distribution in the reservoir is shown at a time instant t0.
Following the sedimentation, the particle-free supernatant liquid is poured off the precipitate or taken out of the reservoir by means of a pipette.
Miniaturized analysis systems on rotating discs offer the simple possibility to sediment particles from suspensions, due to their centrifugal drive. But for further integrated process execution, in most cases the spatial separation of both phases from each other is required. The concluding procedure of pouring out or pipetting off, in particular, is difficult to represent technologically on this integrated microsystem, because the macroscopic method would correspond to a tilt of the channel axis with respect to the direction of the centrifugal force. Due to the further rotation axis, however, this could only be effected with significantly increased instrumental effort.
As it has been mentioned, an important goal with medical diagnostic systems is the integration of complete process chains from the preparation of blood to an analytical result. Various, so-called “lab on a chip” systems have been proposed, see M. J. Madou and G. J. Kellogg, Proc. Of SPIE, vol. 3259, 1998, pages 80-93; G. Thorsen, G. Ekstrand, U. Selditz, S. R. Wallenborg, and P. Andersson, Proc. Of UTAS 2003, eds. M. A. Northrup, K. F. Jenson, D. J. Harrison, Kluwer Academic, 2003, pages 457-460.
Various microfluidic structures on centrifugal platforms are known. These include, among others, sample preparation, flow control by capillary valves and further fluidic networks, see WO 0079285, WO 2004058406, WO 03024598. Furthermore, systems for the separation of blood into plasma and cells in non-centrifugally driven Microsystems are known, see WO 2004074846, WO 2004029221.
WO 2004/061413 A2 concerns a microfluidic apparatus enabling separation of particles in a liquid sample, and particularly of blood into its components, for further analysis. The separation into red blood cells and plasma takes place during few seconds after the blood sample has been introduced into a separation chamber by centrifugal force. A radially passing feed channel, leading into the separation chamber at a radial internal wall, is provided. The separation mechanism here critically depends on surface interactions.
From DE 3723092 C1, a passage centrifuge for industrial production of proteins from human blood plasma is known, in which plastic containers are arranged, which are arranged concentrically with respect to each other in a centrifugal drum, are mutually connected in the bottom region by at least one channel, form annular or ring-segment-shaped chambers, and of which the plastic container or containers facing the rotational axis of the centrifugal drum have an inflow port and the plastic container or containers provided in the outer region of the centrifugal drum have an overhead outflow channel.
In U.S. Pat. No. 4,010,894, a fluid container for use in a centrifugal system for separating the different constituents of blood is described. The container includes two circular layers of flexible material, having center openings. The outer peripheral edges and the inner annular portions around the center opening are connected to each other. Concentrically arranged inner and outer annular channels are formed at the outer peripheral portion of the assembly. Radial arcuate portions are connected to each other, whereby interrupted annular channels are formed. At a first end of the inner annular channel, an inlet tube is provided, which extends outwardly from the center opening and communicates with the first end of the inner annular channel. At the outlet or second end of the inner annular channel, a radially extending interchannel connector is provided, which comprises a sealed portion extending between the adjacent ends of the inner and outer annular channels. At this outlet end of the inner channel, also a radially enlarged region is provided, which acts as a first collecting chamber, into which an outlet tube extending from the inner opening leads. A second outlet chamber is provided at the outlet end of the outer annular channel.